This invention relates to a process for isolating glycomacropeptide (GMP) from a feedstock. More particularly, it relates to the isolation of GMP to a purity under which it has an amino acid composition containing less than 0.5% w/w of phenylalanine (Phe).
GMP is one of a number of names for the peptide split off from xcexa-casein by the enzymes chymosin and/or pepsin. The peptide is also known as casein macropeptide (CM) or casein derived peptide (CDP).
GMP is found in sweet wheys. GMP carries all of the carbohydrate groups of the parent xcexa-casein protein. xcexa-casein is the only casein protein which is glycosylated. Another feature is that when the pH of a solution of GMP is less than 4 the molecular weight is 9000 Da. When the pH is greater than 4, the GMP apparent molecular weight increases to 45000 Da. When concentrating solutions of GMP by ultrafiltration it is preferable for the solution to have a pH greater than 4 unless the membrane cut-off is xe2x89xa610,000 Da. A further feature of GMP is that it does not contain any aromatic amino acids including Phe in its structure.
GMP has a number of potential therapeutic uses as well as having functional properties which make it very useful as an ingredient in food compositions. One important utility is as a nutritional component for use in the diets of persons suffering from phenylketonuria (PKU) (Marshall S, (1991), Food Res Quarterly, 51, 86-91). Phenylketonurics lack Phe hydroxylase in their metabolic system. Therefore, they are unable to utilise Phe present in foods. This can result in a sufficient accumulation of Phe to cause irreversible mental retardation. In order for GMP to be safe for use in feeding to phenylketonurics the Phe level should be as low as possible. A representative product specification would require that the Phe level be 0.5% w/w or less and it would be desirable to have a method of isolating GMP to such a low level of Phe impurity on a production level scale.
Although a number of processes for isolating GMP are known, none of those processes have been shown to be capable of producing GMP having 0.5% w/w or less Phe impurity on a production level scale. One reason for this is that a GMP product of sufficient purity has not yet been manufactured. For example, assuming that the contaminant proteins contain an average 4% Phe, then a purity of greater than 88% GMP is required to give less than 0.5% Phe.
For example, EP A291264 discloses an industrial scale process for the purification of GMP from a mixture of xcex1, xcex2 and xcexa caseins whereby the caseins are subjected to enzymatic milk-coagulating treatment to obtain non-coagulate components as an effluent. The effluent is then kept or rendered acidic to form a precipitate and the remaining effluent subjected to a desalting treatment to produce a GMP with 82% purity. No amino acid analysis data of the purified GMP is disclosed. This process is further described in Tanimoto et al, Biosci. Biotech. Biochem., 56(1), 140-141, 1992, which discloses a large-scale preparation of GMP from rennet casein whey. The whey was filtered and the filtrate desalinated and freeze dried to produce GMP powder having a Phe content of 2.4% w/w. The GMP powder was further purified by Q-Sepharose(copyright) ion-exchange chromatography to give a purified GMP with 0.9% w/w Phe.
GB 2188526 discloses a process for producing a proteinaceous material from milk or casein-containing milk products at pH 4-6 using anion exchange chromatography. No amino acid analysis data of the purified proteinaceous material is disclosed.
GB 2251858 discloses a process for producing GMP from milk raw materials by adjusting the pH of the material to xe2x89xa64, contacting the solution with an anion exchanger, concentrating and desalinating the eluate to give a GMP a 51% purity. This product may be further purified by ultrafiltration according to U.S. Pat. No. 5,075,424 (below) to produce GMP of 87% purity. No amino acid analysis data of the purified GMP is disclosed.
Outinen et al, Milchwissenschaft 50(10), 570-574, 1995, discloses a process for isolating GMP from cheese whey using an inexpensive polystyrenic strong basic anion exchange resin at pH 5. The Phe content of the purified GMP was 0.9% w/w. Further purification of this product by TCA precipitation gave a Phe content of 0.63-0.79% w/w.
U.S. Pat. No. 5,075,424 discloses a process for producing GMP from milk starting materials at pHxe2x89xa64 by ultrafiltration. This process relies on the discovery that GMP has a smaller apparent molecular weight at pH less than 4 than at pH greater than 4 so that only GMP will pass through a 10,000-50,000 molecular weight cut off membrane at pH less than 4. After ultrafiltration, the pH of the permeate is adjusted to pHxe2x89xa74 and subjected to a second ultrafiltration using a membrane with a molecular weight cut-off of xe2x89xa650,000 Da to give a GMP retentate which is 82% pure. No amino acid analysis data of the purified GMP is disclosed.
Kawasaki et al, Milchwissenschaft 48(4), 191-195, 1993 discloses a purified GMP product using the method of U.S. Pat. No. 5,075,424 having a Phe content of 0.6% w/w and a purity of 81%.
Kawasaki et al, Milchwissenschaft 47(11), 688-693, 1992 discloses the same GMP product as above which is fractionated further by analytical chromatography systems (size exclusion chromatology and anion exchange chromatography). The purified GMP products had Phe content of 0.1-0.3%. However, such analytical systems are not suitable for mass production.
U.S. Pat. Nos. 4,042,575 and 4,042,576 each disclose a process of purifying glycoproteins (including GMP) by double ultrafiltration or flocculation of whey proteins followed by ultrafiltration. No amino acid analysis data for the purified glycoproteins is disclosed.
JP 04243898 discloses a process for producing GMP from cheese whey etc at pH 3-6 by heating, adding ethanol, centrifuging and loading the supernatant onto an anion exchange column and eluting GMP with 0.3M ammonium bicarbonate. This process is also reported in J. Dairy Sci 74, 2831-2837, 1991, where amino acid analysis data is given for the isolated GMP. However, Phe is not included in their analysis.
AU 74081/91 discloses a process for producing GMP from a whey protein concentrate in which the proteins are flocculated, the supernatant concentrated by ultrafiltration and the retentate treated with ethanol to produce a precipitate and a second supernatant. The second supernatant is collected and dried to give GMP powder of 84% purity. No amino acid data is disclosed.
JP 3-294299 discloses a process for the manufacture of GMP from whey by heating a 5-50 wt% solution of whey proteins followed by freezing and thawing. The supernatant is then separated, desalted and concentrated by ultrafiltration. No purity or amino acid analysis data of the recovered GMP is given.
WO 94/159252 discloses a method of producing GMP from whey using ultrafiltration, heat treatment of the retentate at 95xc2x0 C. for 15 minutes, adjustment of the pH to 4-5 filtering and collecting GMP from the filtrate. The GMP is 70% pure and the Phe content is ⅓ that of the raw material. The exact concentration of Phe is not disclosed.
EP 0488589 discloses a process for producing GMP by contracting milk raw materials with an ion exchanger, collecting the protein which does not adsorb on the ion exchanger, concentrating and desalting to obtain GMP of 55%-88% purity. No amino acid analysis data is provided.
All of the aforementioned processes are aimed at producing enriched GMP fractions and are not necessarily concerned with reducing Phe content of the GMP as they are not concerned with producing diets for phenylketonurics.
According to WO 93/17587, Phe may be removed from proteinaceous material such as whey protein by enzyme hydrolysis and ultrafiltration to remove unhydrolysed protein. The permeate containing mainly amino acids and small peptides is passed through a column of adsorption resin to remove Phe at pH 6-7 (to give a product with 0.3% Phe) or pH 3-5 (to give a product with xe2x89xa60.1% Phe). Methods involving enzymatic hydrolysis of proteins such as this one cannot be used to purify GMP since they would degrade it to low molecular weight fractions. They are thus not suitable for producing purified GMP.
Smithers W et al. Food Australia 43(6), June 1991, 252-254 outlines a method of isolating GMP (CDP) from cheese or rennet casein whey by selective adsorption at pH 5 using an anion exchanger. Non-bound proteins are then washed out of the exchanger using water and the GMP eluted with mild acid. The eluate is microfiltered to remove a contaminant and subsequently concentrated by ultrafiltration and dried to give a GMP powder stated to have  greater than 90% purity but no Phe analysis data is given.
The present invention provides alternative methods of producing purified GMP having 0.5% w/w or less Phe impurity on a production level scale.
It is therefore an object of this invention to go some way towards achieving this desideratum or at least to offer the public a useful choice.
Accordingly, the invention may be said broadly to consist in a process for the purification of GMP characterised in that the purified GMP has a Phe content of not greater than about 0.5% (w/w) which comprises:
contacting a GMP containing feedstock with a first anion exchanger under conditions to adsorb said GMP,
eluting said adsorbed GMP from said anion exchanger and removing impurities from said GMP containing eluate by either:
i) contacting said GMP containing eluate with a cation exchanger in conditions under which said impurities in said eluate are adsorbed onto said cation exchanger, or
ii) precipitating said impurities in said GMP containing eluate using conditions by means of which said GMP remains in solution, or
iii) contacting said GMP containing eluate with a second anion exchanger in conditions under which said impurities in said eluate are adsorbed onto said anion exchanger and,
recovering said GMP from whichever one or more of said steps (i), (ii) or (iii) was used.
In one alternative said conditions in said first anion exchanger are such that said GMP is adsorbed selectively.
In another alternative said conditions in said first anion exchanger are such that both said GMP and other whey proteins in said feedstock are adsorbed and then said first anion exchanger is eluted under conditions which selectively elute said whey proteins and then elute GMP.
Preferably said condition for eluting said whey proteins or said impurities is obtained by adjusting said first anion exchanger to a pH of 4-5 if necessary and then using an appropriate eluent such as 20-60 mM sodium chloride.
The whey protein in the eluate may optionally be recovered as an additional product.
Preferably the feedstock for said first anionic exchanger is a cheese or rennet whey, an ultrafiltration (UF) retentate or a whey protein concentrate produced from a cheese or rennet whey or a whey protein isolate produced from one of these by an anion exchanger.
Alternatively said feedstock has been pretreated to remove substantially all the whey proteins contained therein except GMP.
Preferably said whey proteins have been removed by either heat treating said feedstock or contacting it with a cation exchanger.
In a further alternative said feedstock is derived from a casein or caseinate product prepared by acid precipitation and has been treated with a suitable enzyme to release GMP in solution and precipitate casein.
In one embodiment said GMP containing eluate from said first anionic exchanger is treated by said alternative (i).
In a second embodiment said GMP containing eluate from said first anionic exchanger is treated with said alternative (ii).
In a third embodiment said GMP containing eluate from said first anionic exchanger is treated with said alternative (iii).
In another alternative said GMP containing eluate from said first anionic exchanger is treated with said alternative (i) followed by treatment with said alternative (ii).
In another alternative said GMP containing eluate from said first anionic exchanger is treated with said alternative (ii) followed by treatment with said alternative (i).
In another alternative said GMP containing eluate from said first anionic exchanger is treated with said alternative (i) followed by treatment with said alternative (iii).
In a still further alternative said GMP containing eluate from said first anionic exchanger is treated with said alternative (iii) followed by treatment with said alternative (i).
Preferably said GMP containing feedstock is contacted with said first anionic exchanger at a pH between 3 and 9.
More preferably, said pH is between 3.5 and 5.
In one alternative said eluent for desorbing said GMP from said first anionic exchanger contains salt in a sufficient quantity to make the solution approximately 20-200 mM and acid in a sufficient quantity to lower the pH to 2-3.
In another alternative said eluent for desorbing said GMP from said first anion exchanger contains salt in a sufficient quantity to make the solution about 175 mM and base in sufficient quantity to raise the pH to about 8 to 9.
Preferably, said GMP containing eluate in alternative (i) is contacted with said cation exchanger at a pH between about 1.5 and about 4.5 and at a salt concentration of up to 100 mM.
Preferably said salt is sodium chloride.
Preferably said GMP is neutralised, concentrated by ultrafiltration and/or diafiltration prior to drying.
Preferably in said alternative (ii) said conditions are pH 3.5-4.5 attained by adding an acid or base to the GMP containing eluate.
Preferably said acid is hydrochloric acid.
Preferably said base is sodium hydroxide.
Preferably after said precipitation step the mixture is centrifuged or microfiltered and the supernatant or permeate is either further processed according to alternative i) or is immediately neutralized, concentrated and dried to produce a GMP powder.
Preferably, in said alternative iii) said second anion exchanger is substituted with amino groups bearing a hydrophobic group. Preferably, said hydrophobic group is selected from the group consisting of C6-C12 hydrocarbon groups including aromatic hydrocarbons. Preferably said hydrophobic group is a C8 hydrocarbon group.
Preferably, the GMP loaded first anion exchanger in the initial step is washed with a dilute salt solution, such as 50 mM sodium chloride, prior to said GMP elution to remove impurities therefrom.
Preferably in said alternative iii), said GMP containing eluate is adjusted to a salt concentration of 125-200 mM with a physiologically acceptable salt and the pH is at least 7.
Preferably the non-adsorbed GMP passing through said second anion exchanger is either further processed according to alternative i) or is immediately neutralised, concentrated by ultrafiltration and/or diafiltration and dried.
In one alternative the GMP from the initial stage or alternative i) is concentrated by ultrafiltration before conducting alternative ii) or iii).
Preferably the process is conducted under conditions which promote a high yield.
In another alternative the invention may be said broadly to consist in a process for the purification of GMP to a Phe content of not greater than about 0.5% (w/w) which comprises:
contacting a GMP feedstock with an anion exchanger under conditions to adsorb selectively said GMP, and
eluting selectively said adsorbed GMP from said anion exchanger leaving impurities on said anion exchanger, and
recovering GMP from the GMP containing eluate.
In this alternative, these conditions are used to selectively desorb said GMP from the anion exchanger leaving impurities on said anion exchanger in a single step by combining the initial step with alternative (iii).
Preferably said GMP loaded first anion exchanger in the initial step is washed with a dilute salt solution, such as 50 mM sodium chloride, prior to said GMP elation to remove impurities therefrom.
Preferably said anion exchanger is regenerated by eluting said impurities therefrom after said GMP elution.
Preferably said GMP is selectively eluted with an eluent having a pH of about 8-9 and a salt concentration of about 175 mM.
The invention may also be said broadly to consist in a method of purifying GMP from a GMP feedstock substantially as herein described with reference to FIG. 1 of the accompanying drawings and by reference to any example thereof.
The invention may also be said broadly to consist in GMP purified to contain no more than 0.5% w/w of Phe whenever prepared by a process as defined herein.
More preferably the invention may be said broadly to consist in GMP purified to contain no more than 0.4% Phe whenever prepared by a process as defined herein.
This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers art mentioned herein which have known equivalents in the are to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
The invention consists in the foregoing and also envisages constructions of which the following gives examples.